An efficiently operating haemostatic system is of vital necessity for the mammalian organism. In the plasma of a healthy organism a dynamic equilibrium exists between the fibrinolytic system and the coagulation system, as a result of which an efficiently operating vascular network is maintained. When vascular lesions occur, the coagulation system deposits a fibrin matrix which, after achieving the haemostatic condition, is broken down again by the fibrinolytic system. In cases in which the fibrinolytic potential of the organism is not sufficient to break down intravascular thrombi that have been formed, for example in patients who suffer from thromboembolisms or post-operative complications, supporting the organism by the administration of thrombolytic agents or anticoagulants proves indispensable.
Anticoagulants like e.g. hirudin, heparin, low molecular weight heparins or low molecular weight synthetic thrombin inhibitors counteract the coagulation system by inhibiting the formation of fibrin clots. Hirudin which has been known for a long time and which occurs naturally in leeches (Hirudo medicinalis) (Walsman, P. and Markwardt, F. (1981) Pharmazie 36, 653) is the strongest thrombin inhibitor of all naturally occurring and synthetic anticoagulants known with a complex dissociation constant of 2.times.10.sup.-14 M, thus preventing the formation of fibrin from its precursor fibrinogen. Other enzymes of the blood coagulation cascade are not inhibited by hirudin. In contrast to heparin which is the preferred anti-coagulant in conventional anticoagulation therapy, hirudin exerts its inhibiting action directly on thrombin and, unlike the former, does not act through antithrombin III. No effect on heart rate, respiration, blood pressure, thrombocyte count, fibrinogen and haemoglobin could be observed after intravenous administration of hirudin to dogs, even in high doses. In tests on rats, pigs and dogs, hirudin has proved effective in experimental thrombosis (induced either by stasis, vascular damage or by the injection of thrombin), in endotoxin shock, and also in DIC (disseminated intravascular coagulation).
Hirudin is not a single polypeptide species but a class of equally acting polypeptides consisting of at least four representatives designated hirudin variant 1 (HV1), hirudin variant 2 (HV2; EP Application 0 158 564), hirudin variant PA (PCT Application WO 86/03493), and "des-(Val).sub.2 -hirudin" (EP Application 0 158 986). The variants differ from each other by a number of amino acids, for example at the N-terminal sequence which is Val-Val-Tyr for HV1, Ile-Thr-Tyr for HV2 and PA and Thr-Tyr for "des-(Val).sub.2 -hirudin". Based on NMR studies, HV1 is composed of an N-terminal core domain with a protruding "finger" (residues 31-36), and an acidic terminal loop (Clore et al., EMBO Journal 6, 529, 1987). All abovementioned hirudin variants have an accumulation of hydrophobic amino acids at the N-terminus and an accumulation of polar amino acids at the C-terminus, a tyrosine residue (Tyr 63) present as sulphate monoester, three disulphide bridges and the anticoagulant activity in common.
Recently, cDNAs and synthetic genes coding for hirudin variants have been cloned and expressed in microbial hosts. Although the expression products lack the sulphate monoester group at Tyr63 - and were therefore designated "desulphatohirudins" - they turned out to exhibit approximately the same biological activity as the natural sulphated hirudins. De. sulphatohirudin variant HV1 has been expressed in Escherichia coli (European Patent Applications No. 158 564 and 168 342) and in Saccharomyces cerevisiae (European Patent Applications No. 168 342, 200 655, 225 633 and 252 854). Similarly, desulphatohirudin HV2 has been expressed in Escherichia coli (European Patent Application No. 200 655, PCT-Application No. 86/01224) and des-(Val)2-desulphatohirudin has been expressed in Escherichia coli (European Patent Application No. 158 986).
The main use of hirudin and other anticoagulants is for prevention or treatment of thrombi in arteries, veins or extracorporal circulation. One prerequisite for the therapeutic application of anticoagulants is the availability of an antidote highly efficient in neutralizing the anti. coagulation activity which can be used in order to survey and regulate the effect of the anticoagulant. So far, such an antidote (protamine sulphate) is available for heparin, which is therefore (despite the occurence of negative side-effects and numerous non-specific reactions) up to now the main anti. thrombotic agent used in hospitals. However, protamine sulphate is ineffective as an antidote to other anticoagulants such as hirudin, which is a much more potent antithrombotic. The reluctance to use hirudin or other anticoagulants instead of heparin in the absence of an efficient antidote could be overcome if there were an antidote available, which would rapidly reverse the anticoagulant effect and reduce the risk of haemorrhages to any patient with higher levels of anticoagulation than those desired.